Protocol 25320: Standard PCR Assay - Tg(Ins2-OVA)
Version 5.2

Notes

The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX). To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility. Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.

Expected Results

Melt Peaks
Wt = 89.7°C +/- 1
Tg = 82.5
°C +/- 1

Transgene = ~475 bp
Internal positive control = 380 bp


JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR6021 CAA GCA CAT CGC AAC CA Mutant Forward A
oIMR6022 GCA ATT GCC TTG TCA GCA T Mutant Reverse A
oIMR6023 CGA GCT CGA GCC TGC CTA TCT TTC AGG TC Wild type Forward A
oIMR6024 CGG GAT CCT AGT TGC AGT AGT TCT CCA G Wild type Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR6021 0.50 uM
oIMR6022 0.50 uM
oIMR6023 0.50 uM
oIMR6024 0.50 uM
Glycerol 6.50 %
Dye 1.00 X
Kapa 2G HS taq polym 0.03 U/ul
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 65.0 -- -0.5 C per cycle decrease
4 68.0 --
5 -- repeat steps 2-4 for 10 cycles (Touchdown)
6 94.0 --
7 60.0 --
8 72.0 --
9 -- repeat steps 6-8 for 28 cycles
10 72.0 --
11 10.0 -- hold
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.

Strains Using This Protocol

Stock Number Strain Name
005432 C57BL/6-Tg(Ins2-OVA)307Wehi/WehiJ
005433 C57BL/6-Tg(Ins2-OVA)59Wehi/WehiJ
005431 C57BL/6-Tg(Ins2-TFRC/OVA)296Wehi/WehiJ
3 strains use this protocol